(1) Field of the Invention
The present invention relates to the field of molecular biology, in particular, the invention is concerned with novel selection genes to be used for improved protein production from transformed expression systems.
(2) Description of Related Art
In recent years the budding yeast Pichia pastoris has become a popular organism for the expression of heterologous proteins of academic and commercial interest (Cereghino et al., Curr. Opin. Biotechnol. 4: 329-332 (2002); Cereghino and Cregg, FEMS Microbiol. Rev. 24: 45-66 (2000). It was recently shown that it is possible to genetically modify the glycosylation machinery of P. pastoris and express heterologous glycoproteins decorated with complex type human glycans (Choi et al., Proc. Natl. Acad. Sci. 100: 5022-5027 (2003); Hamilton et al., Science 301: 1244-1246 (2003); Bobrowicz et al., Glycobiology 14: 757-766 (2004); Hamilton, Science, 313: 1441-1443 (2006). However, a need remains for methods and materials to achieve higher cellular productivity in transformed cell lines, such as transformed P. pastoris cell lines.
Over the years, numerous auxotrophic and dominant selectable markers have been developed (Higgins et al., Methods Mol. Biol. 103: 41-53 (1998); Lin Cereghino et al., Gene 263: 159-169 (2001); Nett and Gerngross, Yeast 20: 1279-1290 (2003); Nett et al., Yeast 22: 295-304 (2005) and used to construct protein expression vectors for various applications. Commonly, a gene of interest is integrated into the P. pastoris genome using a plasmid that is either linearized in the marker gene, another homologous region on the plasmid or in the AOX1 promoter fragment and transformed into the appropriate auxotrophic mutant. Homologous recombination of the free DNA termini then results in single-crossover type integration into these loci. Most P. pastoris transformants will contain a single copy of the expression vector, but to obtain transformants that express a high level of the protein of interest it is often desirable to screen for multi copy integrants. Using expression vectors that contain drug resistance genes as selection markers like KanR or ZeoR it is possible to increase the number of transformants harboring multiple copies of the expression vector by increasing the level of drug used for selection. One significant disadvantage of the single-crossover type integration lies in the fact that the multiple integrated copies can collapse back into a single copy by homologous recombination. This can be especially problematic during scale-up of the expression reaction during fermentation if the protein of interest is toxic to the cells or the eviction of several copies of expression plasmid possesses other growth benefit for the cells.
U.S. Pat. No. 5,584,039 relates to a selectable marker gene ADE2 isolated from Pichia methanolica. Piontek et al., Appl Microbiol. Biotechnol. 50:331-338 (1998) relates to novel gene expression systems in Schwanniomyces occidentalis and Pichia stipitis, which systems utilize vectors containing an ADE2 marker and a putative replication sequence. However, no corresponding gene has previously been isolated from Pichia pastoris, and the effects of transformation with ADE2 in P. pastoris have not previously been identified.
Accordingly, a need exists for improved methods of transformation, selection and expression of heterogeneous genes using the Pichia pastoris yeast as the host expression system.